This collaborative project seeks to gain a deep mechanistic understanding of the Mediator kinases (CDK8 and CDK19) and the CDK8 module. CDK8 and its paralog, CDK19, are considered Mediator kinases because of their stable, but reversible, association with the 26-subunit Mediator complex. Mediator is a global regulator of pol II transcription and appears to be required for expression of perhaps all pol II transcripts, which include all protein-coding and most non-coding RNA genes. CDK8 and CDK19 are linked to a growing number of cancers and developmental diseases, indicative of their essential-yet poorly understood-biological roles. Here, we propose to study CDK8 and CDK19 function in the context of serum response signaling, which is fundamentally important for cell physiology, development, and cancer biology. CDK8 in particular (CDK19 is less studied) has been shown to regulate transcription of serum response genes; however, the mechanisms remain unclear due to inherent limitations with cellular knockdown approaches. Working with our collaborator Dr. Matt Shair (Harvard), we have characterized a small molecule that is an extremely potent and selective Mediator kinase (CDK8 and CDK19) inhibitor. Using this reagent, we can for the first time reliably assess the mechanistic roles of Mediator kinases in human cells. In the context of serum response, we will identify the kinase substrates for CDK8 and CDK19 by using SILAC phosphoproteomics (with/without the Mediator kinase inhibitor). Also during serum response, we will use GRO-Seq to determine how Mediator kinases affect transcription genome-wide. GRO-Seq is uniquely suited to address these questions because, among other reasons, it provides a direct and immediate picture of active transcription (including all classes of transcripts-antisense, divergent, eRNA, lncRNA, tRNA, and so on), which is essential for understanding the rapid (within minutes) cellular response to serum stimulation. CDK8 functions in the context of a four-subunit, 600 kDa CDK8 module that also contains CCNC, MED12, and MED13. Because of its genome-wide association with Mediator, the CDK8 module seems poised to broadly regulate pol II transcription. The structure of the CDK8 module is poorly understood, which limits our understanding of the molecular mechanisms that regulate its essential biological functions. In Aim 2, we seek to define the 3D architecture and identify functionally relevant interfaces within the CDK8 module by applying well-tested crosslinking-mass spectrometry (CXMS) approaches and innovative 3D modeling techniques. These data will be supported by detailed in vitro mechanistic studies and cell-based functional validation assays, with a goal of defining the molecular mechanisms by which the CDK8 module regulates pol II transcription and how its kinase activity is regulated. Collectively, the diverse and complementary set of experiments proposed should yield fundamental insights regarding CDK8 module and Mediator kinase function that should drive the field in new directions.